Much has been published regarding H. pylori which inhabits the human gastric mucosa. It is a gram-negative spiral rod-shaped bacterium having an outer membrane with four to six polar flagella which are sheathed and have bulbous ends; each H. pylori bacterium is about 0.85 .mu.m (micrometer) in diameter with an average length of 2.9 .mu.m. Known pathogenic (disease) factors of H. pylori are (i) urease which is produced by the bacteria to allow it to thrive in a strong acid environment in the range from pH 1-3, (ii) flagella which provide the bacteria with mobility, and (iii) a proteinaceous outer membrane of the cells which membrane helps the cells to stick to the gastric mucosal cells.
To date, treatment to subdue secretion of gastric acid, for example with H2 isolator, is deemed unsatisfactory over the long term due to recrudescence which is now countered with medicines which act directly on the H. pylori. Presently, trends in the fight against infection by H. pylori may be categorized as follows: (a) development of antibiotics showing a direct effect against H. pylori, (b) development of vaccines for H. pylori, and (c) using anti-H. pylori antibodies which allow the live H. pylori to be terminated. For prophylaxis, (b) and (c) are preferred.
Bhatia et al in J. Clin. Microbiol. 27: 2328-2330, 1989, disclosed that L. acidophilus could inhibit the growth of H. pylori in vitro, and that this effect was due to lactic acid. Midolo et al in J. Appl. Bacteriol. 79:475-479, 1995, disclosed that L. casei, L. bulgaricus, Pediococccus pentosaceus and Bifidobacterium bifidus could inhibit the growth of H. pylori in vitro, and that this effect was due to organic acids produced by these bacteria. However, as stated in European Patent Application EP 0 877 032 A1 to Kodama et al (hereafter, "the '032 application"), one cannot expect experiments conducted in vitro to be replicated in the stomach. An attempt to use Lactobacillus salivarius as a probiotic to inhibit growth of H. pylori is reported by Aiba et al in The Meeting of the 30th Japan Germ-free Animal Gnotobiology Society, Program and Abstracts, pp 22, Requested Title 18, "New Attempt for Inhibiting H. pylori" (January 1997). They also used anti-H. pylori antibodies in the yolks of eggs of hens immunized with formalin-killed, whole H. pylori cells. In germ-free mice, the effectiveness of the L. salivwius was 2 to 3 orders of magnitude greater than that of the antibodies; effectiveness in the environment of the stomach of a mammal such as a normal mouse, or in the pH 1-3 of a human, was not investigated. Though the particular strain of L. salivarius was not identified, there is no reason to believe that any lactic acid bacteria will be comparably effective even in a germ-free mouse; data presented below indicate that several species of Lactobacillus show high in vitro activity, but are not as effective in vivo as others with comparably high in vitro activity. In particular, certain strains of L. casei are insubstantially effective in vitro relative to one found to be quite effective both in vitro and in vivo; only the strain HY 2782 lodge in the Korean Culture Collection, Seoul, Republic of Korea under Depository No KFCC-10803 was found sufficiently effective to be useful in a food, as shown in the comparison below.
In the prior art there are taught many immunization schedules under which growth of anti-H. pylori antibodies can be stimulated, most relevant among which are the disclosures of Japanese Patent Application Kokai No. 4-275232 to Takahashi et al, which discloses antibodies obtained in eggs of hens immunized against H. pylori whole cells as an antigen; and, the disclosure of the '032 application which discloses antibodies obtained in eggs of hens immunized against (i) flagella of H. pylori separated from the rest of the cells; and (ii) urease of H. pylori separated from the rest of the cells, these being pathogenic factors associated with H. pylori. Antibodies obtained from either (i) or (ii), by themselves, had no noticeable effect on the number of cells in the stomach of five mice; however, (i) and (ii) in combination eliminated the H. pylori cells from the stomachs of 5 out of 5 mice. (see Table 2 in the '032 application).
Takahashi et al teach the use of a solution of shattered or comminuted H. pylori as antigen, but the beneficial effects are relatively small because the solution additionally contains many other different proteins which appear to dilute, if not diminish or negate, the ability of the antigen to generate effective antibodies.
Furthermore, Kodama et al teach that either the anti-urease antibodies or the anti-flagella antibodies, or both together, may be used in combination with at least one organism selected from the group consisting of lactic acid bacteria, Enterococci, yeasts and Bacillus to inhibit the growth of H. pylori in the stomach, teaching that the presence of any live organism unexpectedly enhances the effectiveness of the antibodies, though the live organism, by itself, was ineffective in the environment of the stomach. In particular, Kodama states that L. acidophilus, L. casei, L. bulgaricus, Pediococcus pentosaceus and Bifidobacterium bifidus were all reported to inhibit growth of H. pylori in vitro purportedly due to organic acids produced by these bacteria, but such effectiveness was of no help to assess the effect in the stomach. Evidence of the synergistic effect of antiurease antibodies and Lactobacillus acidophdus administered in combination orally to H. pylori-infected mice is presented in Table 3 of EP '032. Note however, that only one-half of the population of L. acidophilus is found after 14 days. Without considering the propriety of extrapolating those results to all live organisms tested, it is evident from results presented in Table 3 that one particular strain of L. acidohilus showed a synergistic effect with H. pylori-urease. However, one skilled in the art is unable, without undue experiment-ation, to reproduce the effect reported, because it is not reasonably possible to find the single strain among all the known strains of L. acidophilus which produces the synergistic result.
Confirmation of the ineffectiveness of the live organism, by itself, is stated as follows: "L. acidophilus alone was almost the same as that of the control group, and there was no significant difference between the two groups, as shown in Table 3. Also, gastritis conditions were observed and L. acidophilus had no efficacy on suppressing gastritis." (see page 10, lines 49-52). The tests were performed on hairless mice (NS:Hr/ICR) having a high sensitivity to H. pylori infection. Such mice do not have the normal flora found in a BALB/c mouse which provides a better comparison with a human stomach.
Contrary to Kodama's teaching, we found that to get the beneficial effects of a bacteria in vivo in the stomach, it is critical that we use a live bacteria which by itself is highly effective in vitro against H. pylori--and to boost its effect, to use antibodies produced by antigens of fractionated H. pylori. The term "fractionated H. pylori" refers to particular portions of H. pylori which portions are separated from the remainder of the cells; the separated portions are as follows: (i) urease; (ii) the outer membrane and (iii) the flagella; the remainder of the cells is discarded. Since it is not practical to conduct a very large number of in vivo experiments with H. pylori-infected mice using strains of various bacteria, we chose to use a combination of the three strains found after screening a limited number of strains set forth in Table 1 below. It is recognized that there may be one or more specific strains of L. acidophilus, not suggested in the '032 application, any one of which, by itself, may be effective against H. pylori not only in vivo but also in vitro; they chose to use a strain which was effective in neither.
It is now evident that, in the prior art, the problem of attacking the H. pylori in a stomach relied upon the H. pylori-antibodies collected from one or more constituents of fractionated H. pylori; or, in combination with a bacteria such as L. acidophilus used in the '032 application, or any other bacteria which by itself had no noticeable effect in the environment of a stomach. The invention described hereunder derived from the notion that some bacteria ingested by humans might survive the environment of the stomach for long enough to find the relatively less acidic zones around H. pylori attached in the stomach's lining, and excrete bacteriocins which would attack the H. pylori. A search was made for those bacteria which could be relied upon to provide a major portion of the desired attack, their effectiveness being supplemented with conventionally derived H. pylori-antibodies.
Further, the prior art typically obtained egg yolk powder by freeze-drying an aqueous solution, and was unconcerned with formulating a commercially marketable food fortified with egg yolk powder containing H. pylori-antibodies; this led to a lack of concern to stabilize the antibodies during spray-drying of egg yolk solution to make the powder, spray-drying to sterilize being the preferred commercial method of production of egg yolk powder. Sterilization requires spray-drying at a temperature of at least 65.degree. C. at which temperature the antibodies are unstable. The prior art did not provide a solution to the problem of finding a comestible, non-toxic water-soluble food ingredient which is able to stabilize the antibodies in the egg yolk solution at a pH and temperature which would not deleteriously affect the antibodies.
Despite the development of several medicines for the treatment of disorders due to H. pylori, the prior art has failed to suggest any logical basis for selecting an active strain of non-toxic, live bacteria for such treatment, except trial and error. By "active strain" is meant a non-toxic strain of live bacteria which effectively kills or inhibits the growth of H. pylori grown as a lawn in a growth-conducive anaerobic or microaerophilic environment, on a medium in vitro, in an amount sufficient to provide a zone free of H. pylori, which zone is visually observable with the naked eye. Particularly because it is not possible to predict whether an active strain which produces the appropriate bacteriocins will survive long enough to be effective in the environment of a human stomach, we chose to study lactic acid bacteria, and closely related bacteria, which are beneficial and known to survive in the stomach of a human body; and, if specific strains of those bacteria produced the appropriate H. pylori-specific bacteriocins, sought to deliver the bacteria in food routinely consumed by humans in everyday life.